RESEARCH PAPER

Fully automated dried blood spot sample preparationenables the detection of lower molecular mass peptideand non-peptide doping agents by means of LC-HRMS

Tobias Lange1& Andreas Thomas1 & Katja Walpurgis1 & Mario Thevis1,2

Received: 10 December 2019 /Revised: 26 March 2020 /Accepted: 31 March 2020# The Author(s) 2020

AbstractThe added value of dried blood spot (DBS) samples complementing the information obtained from commonly routine dopingcontrol matrices is continuously increasing in sports drug testing. In this project, a robotic-assisted non-destructive hematocritmeasurement from dried blood spots by near-infrared spectroscopy followed by a fully automated sample preparation includingstrong cation exchange solid-phase extraction and evaporation enabled the detection of 46 lower molecular mass (< 2 kDa)peptide and non-peptide drugs and drug candidates by means of LC-HRMS. The target analytes included, amongst others,agonists of the gonadotropin-releasing hormone receptor, the ghrelin receptor, the human growth hormone receptor, and theantidiuretic hormone receptor. Furthermore, several glycine derivatives of growth hormone–releasing peptides (GHRPs), argu-ably designed to undermine current anti-doping testing approaches, were implemented to the presented detection method. Theinitial testing assay was validated according to the World Anti-Doping Agency guidelines with estimated LODs between 0.5 and20 ng/mL. As a proof of concept, authentic post-administration specimens containing GHRP-2 and GHRP-6 were successfullyanalyzed. Furthermore, DBS obtained from a sampling device operating with microneedles for blood collection from the upperarm were analyzed and the matrix was cross-validated for selected parameters. The introduction of the hematocrit measurementmethod can be of great value for doping analysis as it allows for quantitative DBS applications by managing the well-recognized“hematocrit effect.”

Keywords Dried blood spots (DBS) . Hematocrit (Hct) . Growth hormone–releasing peptides (GHRP) . TAP blood collectiondevice . Doping . Sport

Introduction

The use of prohibited peptidic drugs and non-peptide mi-metics of lower molecular mass (< 2 kDa) to illegally increaseperformance in professional sports has been in the focus ofpreventive doping research for almost a decade. In 2010, the

first LC-MS detection method for growth hormone–releasingpeptide-2 (GHRP-2) in human urine was described [1] follow-ing the identification of the peptide in a nutritional supplement[2]. Subsequently, detection methods were developed and theanalytical spectrumwas continuously expanded.While initial-ly mainly SPE extracts from urine samples were used for thedetection of lower molecular mass peptides by anti-dopinglaboratories [1, 3–9], more recent approaches include a totalof 21–36 target peptides or their metabolites which can bedirectly detected from urine by LC-HRMS (“dilute-and-in-ject”) [10–12]. The growing list of analytes comprises ago-nists of the ghrelin receptor (e.g. the GHRPs), thegonadotropin-releasing hormone (GnRH) receptor, the humangrowth hormone (hGh) receptor, and the antidiuretic hormone(ADH) receptor. As they act on different biological axes, theirvariety of performance-enhancing effects range from fat loss,bone formation, muscle and blood vessel growth to themasking of prohibited substances [4, 13]. Since the World

Electronic supplementary material The online version of this article(https://doi.org/10.1007/s00216-020-02634-4) contains supplementarymaterial, which is available to authorized users.

* Mario Thevism.thevis@biochem.dshs-koeln.de

1 Center for Preventive Doping Research – Institute of Biochemistry,German Sport University Cologne, Am Sportpark Müngersdorf 6,50933 Cologne, Germany

2 European Monitoring Center for Emerging Doping Agents(EuMoCEDA), Cologne/Bonn, Germany

Analytical and Bioanalytical Chemistryhttps://doi.org/10.1007/s00216-020-02634-4

Anti-Doping Agency (WADA) listed the GHRFs (growthhormone–releasing factors) including growth hormone secre-tagogues (GHS) and GHRPs in 2013 under section S2 “pep-tide hormones, growth factors, related substances, and mi-metics”, several adverse analytical findings (AAFs) were re-ported, mostly from strength sports, which can be attributed tothe anabolic effects of these drugs. From 2016 to 2017, thenumber of GHRF testing was increased by 17% [14].Recently, glycine-modified analogues of GHRP-2, GHRP-6,and ipamorelin were identified in seized material [15–17]. Allof these compounds are classified as non-threshold substancesand are prohibited by WADA at all times [18] (in- and out-of-competition).

Urine has been the preferred matrix for the detection ofthese lower molecular mass peptides in routine doping con-trols as most analytes demonstrated sufficient stability inurine. Pharmacokinetic studies have demonstrated rapid elim-ination rates for GHRP-2 [19] and GHRP-6 [20] from bloodwith a biological half-life of 2.5 ± 1.1 h for GHRP-6. Afterintravenous (i.v.) administration of GHRP-2, detection timesappeared shorter in serum than in urine samples, but forGHRP-6, detection windows were found to be comparablein both matrices [21, 22]. Nevertheless, for urine analysis,knowledge about the metabolic fate of peptide drugs is desir-able as the presence of metabolites alongside the intact andunmodified drug (candidate) was shown in the past [3, 9, 23].For example, GHRP-1 and alexamorelin are rapidly degradedand are traceable in urine only with significantly inferior sen-sitivity when compared with their metabolites [11].

Dried blood spots (DBS) represent an alternative matrix,which was found to be minimally invasive, cost-efficient, andanalyte-stabilizing. Furthermore, sample preparation and anal-ysis were automatable [24, 25] with the prospect of effectivehigh-throughput testing. However, the limited sample volumeof 10–20 μL blood, obtained, e.g., from the fingertip, and thehighly complex matrix including hemolyzed blood cells and ahigh content of soluble and insoluble proteins pose a challeng-ing task for sports drug testing laboratories. This suggests theuse of modern chromatographic-mass spectrometric instru-mentation, preferably in combination with an automatedDBS sample preparation workflow, to enable testing for phys-iologically relevant concentration levels for these compoundswithout extensive manual sample preparation such as affinityenrichment. Another aspect to consider in DBS analysis is the“hematocrit (Hct) effect” [26, 27]. The influence of blooddispersal on the DBS filter paper was described to result in aHct-dependent bias in quantitative assays, and the determina-tion of Hct in DBSmay contribute to overcome this limitation.

The aim of this study was to develop and optimize a fullyautomated DBS sample preparation as a multi-analyte initialtesting approach for 46 lower molecular mass peptide andnon-peptide agonists. The subsequent LC-HRMS detectionmethod was validated according to WADA guidelines and

reconstructed post-administration DBS samples containingGHRP-2 and GHRP-6 were successfully analyzed for proof-of-concept. Moreover, an upstream near-infrared (NIR) spec-troscopic measurement was envisaged to support the non-destructive Hct determination before starting with the sampleextraction as described by Oostendorp et al. [28]. In the con-text of anti-doping research, fully automated determination ofsmall molecules from DBS was already achieved as, for ex-ample, for nicotine and adipoRon (a synthetic adiponectinreceptor agonist) with LODs of 5 ng/mL [24, 25].

Materials and methods

Chemicals and materials

Ammonium hydroxide, acetonitrile, acetic acid, methanol,and MiniPax® absorbent packets were obtained from Merck(Darmstadt, Germany). Albumin solution 20% (v/v) was pur-chased from Carl Roth (Karlsruhe, Germany). Formic acidwas bought from Thermo Fisher Scientific (Bremen,Germany), dimethyl sulfoxide (DMSO) was supplied byAlfa Aesar (Haverhill, MA, USA), and Whatman™ FTA®DMPK-C sample collection cards were obtained from GEHealthcare (Uppsala, Sweden). For blood collection fromthe upper arm, “TAP” microneedle-based devices were pur-chased from Seventh Sense Biosystems (Cambridge, MA,USA), and for blood collection from the finger, a Microlet 2lancing device with lancets from Bayer AG (Leverkusen,Germany) was used. The GHRP-2 metabolite and d3-Ala-GHRP-2 metabolite (ISTD 2) were in-house synthesized asdescribed elsewhere [3]. A total of 47 peptide and non-peptidecompounds, including 45 analytes and 2 internal standards(ISTDs), were purchased from different suppliers: Auspep(Melbourne, Australia), Bachem (Bubendorf, Switzerland),BMFZ (Düsseldorf, Germany), Centic Biotec (Heidelberg,Germany), Genscript (Piscataway, NJ, USA), MedChemExpress (Princeton, NJ, USA), Pepscan (Lelystad,Netherlands), Prospec (Rehovot, Israel), Sigma Aldrich (St.Louis, MO, USA), Sanofi (Paris, France) and TorontoResearch Chemicals (North York, ON, Canada). The refer-ence material had a specified purity between 90 and 99%and a specified peptide content between 60 and 94% (seeElectronic Supplementary Material (ESM) Table S1).

Standard solutions

Standard and ISTD stock solutions of the peptides were pre-pared in Milli-Q water with 10% acetonitrile, 2% acetic acid,and 0.5% albumin (v/v) in LoBind tubes. For peptides thatwere hardly soluble, more acetic acid was added (GHRP-1met.: 6%, Gly-GHRP-4: 3%, Gly-GHRP-5: 6%). The stocksolutions had concentrations between 0.5 and 1 mg/mL and

T. Lange et al.

were stored at − 20 °C. A standard stockmix of all compoundswas prepared by diluting the stock solutions to 10 μg/mL (−20 °C). Working solutions of the analytes (25–1000 ng/mL)and ISTD (100 ng/mL) were freshly prepared with the solventmixture used to prepare the aforementioned stock solutions.

DBS sampling methods

EDTA-stabilized blood samples from healthy volunteers wereused as a matrix for the preparation of DBS during methoddevelopment, optimization, and validation. The blood wasfortified with the desired concentration of the peptide standardand mixed briefly before spotting 20 μL onto a DBS card. Todemonstrate specificity and identification capability, capillarywhole blood from the fingertip (by micro lancet) or upper arm(by “TAP” blood collection device) was obtained from fivefemale and five male volunteers. After pricking the fingertip,the first drop of blood was wiped off and then 20 μL weretaken with a pipette and placed onto a DBS card. Themicroneedle-based “TAP” blood collection device combinescapillary action and vacuum extraction to collect capillaryblood from the upper arm into a Li-heparin coated chamber(100 μL) [29]. After several minutes, the completion of theblood collection is displayed by a blood indicator window. Apipette was used to transfer 20 μL from the device onto thefilter paper. Unless otherwise stated, DBS cards were dried for2 h at room temperature (RT) and then stored overnight at 4 °Cin plastic bags with desiccant.

Post-administration samples

From a previous application study, EDTA plasma sampleswere available from a male subject (59 years, 78 kg) [30].Here, a single injection containing 666 μg of GHRP-2 and200 μg of GHRP-6 (Hallandale Pharmacy, FL, USA) wasadministered subcutaneously. Serum samples were collectedafter 30, 90, and 270 min and stored at − 20 °C. For thepreparation of artificial DBS specimens, the serum was mixedcarefully with fresh blood cells (obtained from EDTA-stabilized venous blood) up to a Hct of 40%.

Fully automated measurement of Hct and samplepreparation

The determination of the Hct was realized using a NIRFlex N-500 spectrometer equipped with a fiber optics solids cell fromBüchi Labortechnik AG (Essen, Germany) connected to anautomated DBS sample preparation system from Gerstel(Mülheim an der Ruhr, Germany). Before initiating sequencedmeasurements, an internal reference spectrum was recordedby an internal calibration based on a NIR model designed byOostendorp et al. [28] using 261 patient DBS samples (EDTAfull blood) with different Hct, age, and sex. After every 10

measurements, a white reference cap was placed manually infront of the fiber optic probe in order to perform a whitebalance (external calibration). The calibrations were per-formed according to the manufacturer’s advice. DBS cardswere automatically moved in front of the optic probe tip inorder to non-destructively measure the Hct within 2–3 s.Robotic-assisted sample preparation was then accomplishedby a dual-head multi-purpose sampler (MPS) interfaced with aDBS autosampler (DBSA), a solid-phase extraction (SPE)module loaded with strong cation exchange (SCX) polymercartridges, a multi-position evaporation station (mVAP), and ahigh-pressure dispenser pump (HPD). The devices were con-trolled by the Gerstel Maestro 1 software (version 1.4.49.8)and NIRWare (version 1.5.3000).

LC-HRMS/MS

LC-HRMS/MS analysis was accomplished using a VanquishUHPLC system coupled to a Q Exactive™ HF-X HybridQuadrupole-Orbitrap™ mass spectrometer, both fromThermo Fisher Scientific (Bremen, Germany) with nitrogenas source/collision gas (CMC, Eschborn, Germany). APoroshell 120 EC-C8 analytical column, 3.0 × 50 mm,2.7 μm PS from Agilent Technologies (Santa Clara, CA,USA) separated the analytes chromatographically with 0.1%formic acid as solvent A and acetonitrile, 0.1% formic acid,and 1% DMSO as solvent B, with a flow rate of 350 μL/min.After the injection of 10–20 μL of the sample into the instru-ment, the chromatographic run with an overall runtime of15 min was as follows: 1–40% B over 10 min, 40–90% B in0.5 min, 90% B over 1.5 min, and 1% B for 3 min. Thetemperature of the sampler was set to 10 °C, the column com-partment to 30 °C and the transfer capillary to 320 °C. The ionsource was operated in positive mode with an ionization volt-age of 3.3 kV. TheMS analysis comprised alternating full scanMS experiments with a scan range from m/z 300 to 1500 andtargeted-SIM (t-SIM)/dd-MS2 experiments with an inclusionlist of 53 ions. The resolution (FWHMatm/z = 200) was set to60,000 (full MS), 45,000 (t-SIM), and 15,000 (dd-MS2), au-tomatic gain control target value to 3e6 (full MS), 2e5 (t-SIM),and 5e5 (dd-MS2), and maximum ion injection time to 200 ms(full MS), 25 ms (t-SIM), and 50 ms (dd-MS2), respectively.The t-SIM experiments were acquired with a retention timewindow of ± 0.5 min around the expected elution time of eachanalyte, with an isolation window of 3.0 m/z, and an offset of1.0 m/z. A total of five scan events with a maximum numberof five multiplexed ions were acquired before the next full MSstarted. The normalized collision energy was 35%. The instru-ment was operated by Thermo Scientific Xcalibur, version4.1.31.9.

The t-SIM experiments were used to identify the sub-stances by their precursor ions. Full scan MS experimentswere acquired alternately with t-SIM/dd-MS2 experiments;

Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide...

thus enabling a retrospective data analysis for the detection offormerly unknown substances and metabolites by their pre-cursor ions. The dd-MS2 data provided additional informationto confirm the selected ion signals of the inclusion listHowever, unknown substances cannot be additionally identi-fied via MS/MS within this method.

Method validation of the initial testing procedure

The robotic-assisted DBS sample preparation with sub-sequent LC-HRMS detection for 46 analytes (Table 1)was validated according to WADA guidelines for thevalidation of initial testing procedures (ITPs) for non-threshold substances [31]. Each analyte was identifiedby t-SIM experiment at its retention time using twosignals, referred to as target ion and confirming ion.The signals are isotopes of the respective precursor ionsof the dominant charge state. Gly-GHRP-2 was an ex-ception, as isotope signals of different charge states 1+and 2+ were used for identification. Five male and fivefemale volunteers were chosen to demonstrate specifici-ty and identification capability. For the ‘identificationcapability’, the blood spots prepared from capillary fin-ger or capillary upper arm blood were allowed to dryon the filter paper for 30 min before 4 μL of a 100 ng/mL standard mix was added onto the middle of the spotto obtain a concentration of 20 ng/mL. Varying Hctvalues (24–44%) from different individuals of theseDBS samples were used to prove the assay’s robustnesstowards extractability-related issues. Since differentlimits of detection (LODs) were expected for the indi-vidual analytes, six sample replicates at different con-centrations (0.5, 1, 2, 5, 10 and 20 ng/mL) were pre-pared. The precision was estimated using six samplereplicates each at 20 ng/mL, 50 ng/mL, and 100 ng/mL and the coefficient of variation (CV) of the ISTD-normalized peak areas was calculated. In order to studylinearity, a series of standards within the working rangewith 2, 5, 10, 20, 50, and 100 ng/mL was prepared andthe ISTD-normalized peak areas were analyzed assum-ing a linear (1st order) regression. The analyte concen-trations of the DBS for the validation parameters“LOD,” “linearity,” “precision,” and “carryover” wereprepared by carefully mixing venous EDTA-blood withan appropriate amount of the standard mix (volume ≤5% of the total volume) before spotting onto the DBScard. The recovery was estimated by comparing sixsamples containing 20 ng/mL of the standard mix(pre-extraction) and six blank samples that were forti-fied with 20 ng/mL of all target analytes after the evap-oration (post-extraction). The pre-extraction DBS sam-ples were prepared by adding 4 μL of a 100 ng/mLstandard mix onto an already dried 20 μL EDTA-

blood spot (in the same way as for ‘identification capa-bility’). Analytes were thus located in the center of thespot, which allowed their complete extraction by meansof the 6-mm clamp. Prior to LC-MS analysis, pre-extraction samples were fortified with 4 μL ddH20 andpost-extraction samples were fortified with 4 μL of a100 ng/mL standard mix and mixed briefly. Three blanksamples and three neat samples each fortified with20 ng/mL immediately before the LC-MS analysis wereanalyzed to determine absolute matrix effects. The sta-bility of the analytes on the DBS cards as well as thestability of the Hct values was investigated with respectto different storage times (1, 2, 3, 7, 14, 21 days) andtemperatures (− 20 °C, 4 °C, 20 °C). Two DBS samplereplicates were prepared for each storage condition. LC-MS carryover was determined by analyzing a negativecontrol sample (same matrix) immediately after a sam-ple containing a high analyte concentration of 100 ng/mL. To further study the DBSA-SPE carryover, a blanksample was extracted after this sample. For the specific-ity and identification capability, a cross-validation forDBS obtained from the upper arm (“TAP” device) fromthe same ten volunteers was realized.

Results and discussion

Method development and optimization

The fully automated sample preparation was optimizedwith regard to the extraction agent, the employed ex-traction volume, the SPE purification, and the duration,temperature, and vacuum of the evaporation step. Dueto the good water solubility of the analytes, an aqueoussolution was used for the extraction from the DBS card.Different stationary phases (CN, C12, C8, C18, SCX,strong hydrophobic, general-purpose, and mixed-modecation/anion exchange) were tested for solid-phase ex-traction and ion exchange cartridges, especially SCX,yielded the best results.

Briefly, a DBS was extracted with 1.5 mL ddH2O(4 mL/min, 100 °C) through a clamp with a diameterof 6 mm. Sixty microliters of the deuterated ISTD mix(100 ng/mL) were automatically added through a sepa-rate loop and the sample extract was loaded onto a pre-conditioned SCX SPE. The SCX cartridge was washedwith 2% formic acid and analytes were eluted with1.4 mL of 5% ammonium hydroxide in methanol intoa glass vial. Then, the sample eluate was evaporated inthe mVAP for 37 min at 50 °C and 250 rpm withramping pressure from 200 to 60 mbar. Finally, thesample containing 100 μL could be transferred manual-ly to the LC-HRMS system.

T. Lange et al.

With a cold system start, the total time for a batch of 6samples (maximum number of positions in the mVAP) wasapproximately 2 h. By programming a nesting of indi-vidual sample preparation steps, the total time of

various batches could be significantly reduced. Moredetails about the individual steps and information aboutvolumes, flow velocities, pressure, temperature, and ag-itation can be found in the ESM.

Table 1 LC-HRMS related characteristics and categories of the target compounds

Compound Pre-dominant charge state Target ion [m/z] Confirming ion [m/z] RT [min] Category

Alarelin 2+ 584.3065 584.8080 7.01 GnRH receptor agonistAlexamorelin 2+ 479.7560 480.2574 7.42 Ghrelin receptor agonistAlexamorelin (3–6) met. 1+ 623.2957 624.3030 10.35 Ghrelin receptor agonistAnamorelin 1+ 547.3391 548.3421 11.08 Ghrelin receptor agonistAOD9604 2+ 907.9375 908.4388 6.98 hGH receptor agonistAOD9604 (7–16) met. 2+ 521.7077 522.2092 5.27 hGH receptor agonistBuserelin 2+ 620.3353 620.8367 8.50 GnRH receptor agonist(d3)-Ala-GHRP-2 met. (ISTD) 1+ 361.1948 362.1979 7.68 Ghrelin receptor agonist(d4)-Ala-GHRP-4 (ISTD) 1+ 612.3231 613.3262 9.54 Ghrelin receptor agonistDeslorelin 2+ 641.8276 642.3291 8.54 GnRH receptor agonistDesmopressin 1+ 1069.4342 1070.4370 7.04 ADH receptor agonistFelypressin 2+ 520.7257 521.2271 6.16 ADH receptor agonistFertirelin 2+ 577.2987 577.8001 6.81 GnRH receptor agonistGHRP-1 2+ 478.2505 478.7520 7.65 Ghrelin receptor agonistGHRP-1 (3–6) met. 1+ 620.2883 621.2913 11.02 Ghrelin receptor agonistGHRP-2 2+ 409.7210 410.7240 8.88 Ghrelin receptor agonistGHRP-2 (1–3) met. 1+ 358.1761 359.1792 7.69 Ghrelin receptor agonistGHRP-3 1+ 655.4038 656.4067 6.03 Ghrelin receptor agonistGHRP-4 1+ 608.2980 609.3010 9.74 Ghrelin receptor agonistGHRP-5 1+ 771.3613 772.3643 10.27 Ghrelin receptor agonistGHRP-6 2+ 437.2296 437.7312 6.81 Ghrelin receptor agonistGHRP-6 (2–5) met. 1+ 609.2820 610.2850 10.19 Ghrelin receptor agonistGly-Alexamorelin 2+ 508.2667 508.7681 7.08 Ghrelin receptor agonistGly-GHRP-1 2+ 506.7612 507.2627 7.71 Ghrelin receptor agonistGly-GHRP-2 2+ 438.7330 876.4592 8.93 Ghrelin receptor agonistGly-GHRP-3 1+ 712.4253 713.4281 6.24 Ghrelin receptor agonistGly-GHRP-4 1+ 665.3194 666.3224 9.77 Ghrelin receptor agonistGly-GHRP-5 1+ 828.3828 829.3858 10.52 Ghrelin receptor agonistGly-GHRP-6 2+ 465.7403 466.2417 6.91 Ghrelin receptor agonistGly-Hexarelin 2+ 472.7481 473.2496 6.98 Ghrelin receptor agonistGly-Ipamorelin 2+ 385.2108 385.7123 6.79 Ghrelin receptor agonistGoserelin 2+ 635.3280 635.8294 8.17 GnRH receptor agonistHexarelin 2+ 444.2374 444.7388 6.94 Ghrelin receptor agonistHexarelin (1–3) met. 1+ 427.2088 428.2117 4.87 Ghrelin receptor agonistHistrelin 2+ 662.3409 662.8423 6.92 GnRH receptor agonistIbutamoren 1+ 529.2479 530.2505 10.38 Ghrelin receptor agonistIpamorelin 2+ 356.7001 357.2016 5.77 Ghrelin receptor agonistIpamorelin (1–4) met. 1+ 585.2820 586.2850 7.65 Ghrelin receptor agonistLecirelin (dalmarelin) 2+ 605.3300 605.8314 8.40 GnRH receptor agonistLeuprolide 2+ 605.3300 605.8314 8.16 GnRH receptor agonistLeuprolide (1–3) met. 1+ 453.1881 454.1910 5.27 GnRH receptor agonistLHRH 2+ 591.7938 592.2953 6.39 GnRH receptor agonist[Lys8]-Vasopressin (ISTD) 2+ 528.7231 529.2248 5.02 ADH receptor agonistNafarelin 2+ 661.8251 662.8279 9.36 GnRH receptor agonistNafarelin (5–10) met. 2+ 401.2242 401.7257 8.75 GnRH receptor agonistPeforelin 2+ 630.2889 630.7903 5.47 GnRH receptor agonistTabimorelin 1+ 529.3173 530.3205 10.31 Ghrelin receptor agonistTB500 2+ 445.2531 445.7546 3.61 Synthetic version of an active

region of thymosin β4

Triptorelin 2+ 656.3227 656.8241 8.21 GnRH receptor agonist

Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide...

Method characterization and validation

As shown in Table 1, a total of 46 target analytes were includ-edwithin this study, comprising “classical” peptide drugs suchas ipamorelin (pentapeptide) or goserelin (decapeptide) andnon-peptide drugs (mimetics) such as anamorelin as well asseveral potentially performance-enhancing Gly-derivatives ofthe GHRPs. Moreover, GHRP-1, which was stable in urineonly as its metabolite [10], was successfully analyzed in DBS.Felypressin, a new vasoconstrictor related to vasopressin, wasdetermined for the first time.

The lower molecular mass peptides < 2 kDa were observedto predominantly form doubly-charged molecules under thechosen conditions. However, as already described by others,DMSO as an additive in the LC solvent does not only improvethe ionization efficiency [32] but also influences the chargestate distribution of the peptides, shifting the equilibrium tothe direction of lower charge states [33]. Therefore, a relative-ly large number of 17 singly charged molecules comparedwith 29 doubly charged species were utilized for an unambig-uous identification (target ion) within this assay (t-SIMexperiments).

The assay was characterized by a homogeneous chromato-graphic distribution of the analytes with most substances elut-ing between 5 and 11 min.

The ITP was validated according to WADA’s internationalstandard for laboratories 10.0 [31] (Table 2). As the analytesare non-threshold substances, the minimum criteria for LC-MS confirmation of the identity of analytes are applied todemonstrate the presence of a prohibited substance. The se-lectivity for the 46 substances was demonstrated by analyzing10 blank samples collected from different individuals (maleand female). As exemplarily shown in Fig. 1 (dashed line) andESM Fig. S1, no interfering signals were detected in the blanksamples (specificity). Subsequently, 10 samples from thesame volunteers were fortified with 20 ng/mL of the standardmix and analyzed again (Fig. 1, solid line). Hereafter, all sub-stances of interest could be unambiguously identified (identi-fication capability). The LC conditions were very suitable forthe efficient separation of the analytes with the only exceptionof tabimorelin (poor peak shape). In case of a suspicious find-ing, adapted LC conditions can be used for a “confirmationprocedure” for tabimorelin. In the ion chromatogram at RT =7.3 min, a second peak of hexarelin with an identical mass wasobserved that originated from alexamorelin metabolite (2–7)-NH2. Due to an intensive degradation of alexamorelin inblood as was shown by others [23, 34], several metabolitescan be formed. This metabolite was not included for initialtesting purposes; however, it may assist the confirmation ofan alexamorelin finding. Following this, the alternative “TAP”sampling method for the upper arm was tested and cross-validated for these parameters. In the same way as for theblood collected from the fingertip, the assay’s selectivity was

shown for all target analytes. Furthermore, the LOD was esti-mated by applying a signal-to-noise ratio > 3 for the individualtarget and confirming ions with the following results: 7analytes at 0.5 ng/mL, 7 analytes at 1 ng/mL, 12 analytes at2 ng/mL, 10 analytes at 5 ng/mL, 9 analytes at 10 ng/mL, and1 analyte at 20 ng/mL.

For a more comprehensive assay characterization, addi-tional parameters were determined as, for example, requiredfor confirmation procedures. The individually variable Hctvalues of the DBS samples from a finger prick determinedby the NIR spectrometer ranged between 24 and 40%. Noimpact on the LC-HRMS/MS identification of the analyteswas observed, and the method’s robustness was demonstrated.Some articles reported on a Hct-dependent bias concerningquantitative bioanalysis using DBS, especially when a smallpunch of the DBS was excised [27]. Such phenomena werenot observed in the present study, attributed to the fact thatalmost the entire spot was extracted using the 6 mm clamp,and the issue of nonhomogeneous analyte distribution de-scribed before was therefore negligible. However, within thisassay, the robustness for post-fortified DBS samples (fromblank samples with different Hct) was studied, since thesmallest volume of non-coagulated capillary blood that couldbe obtained from the fingertip was not suitable for mixing withthe standard before spotting onto the DBS card. Due to thisway of DBS sample preparation, in which the analytes werepipetted onto the DBS card, the analytes were rather localizedin the center of the spot. Thus, the impact of e.g. differentialspreading of blood with different Hct was not evaluated here.The carryover after extracting a sample with a high concen-tration of 100 ng/mL was determined for both the DBSA andthe LC-HRMS/MS system and was from 0 to 18.9% (data notshown) and from 0 to 9.9%, respectively. Despite the lowprobability of such highly concentrated doping control sam-ples, it is recommended to rinse the DBSA system thoroughlyon a regular basis to remove any potential residues of bloodcell components and proteins. The total recovery of the meth-od for the different analytes varied between 3.7 and 69.6%,and the matrix effects ranged from 33 to 156%. Values <100% indicate ionization suppression effects and values >100% indicate ionization enhancement effects caused by thesample matrix [35]. The linearity was determined from theLOD to 100 ng/mL or not lower than 2 ng/mL for analyteswith LOD < 2 ng/mL and yielded coefficients of correlation rbetween 0.9862 and 0.9999. For the linear regression, slopeand intercept were additionally specified (ESMTable S2). Theprecision was estimated for 6 replicates per analyte at 20 ng/mL, 50 ng/mL, and 100 ng/mL and was found to be below25% for most substances. All analytes remained stable on theDBS card at all storage conditions over 3 weeks and wererepeatedly identified at a concentration of 20 ng/mL for 2replicates each. The variations of the observed ISTD-normalized peak areas were found to be within the range of

T. Lange et al.

Table2

Mainresults

ofvalid

ation

Com

pound

LOD

[ng/mL]

(n=6)

Linearity

(R)

LOD-100

ng/m

L(n=1)

Precision

at20

ng/m

L[%

](n=6)

Precision

at50

ng/m

L[%

](n=6)

Precision

at100ng/m

L[%

](n=6)

Recoveryat

20ng/m

L[%

](n=6)

Carryover

after

100ng/m

L[%

](n=1)

Matrix

effects

[%](n=3)

Alarelin

50.9991

14.9

17.3

15.4

24.6

0.1

67

Alexamorelin

10.9967

16.8

17.5

7.8

9.9

8.5

113

Alexamorelin

(3–6)

met.

100.9979

31.2

35.3

21.9

13.0

0.1

49

Anamorelin

0.5

0.9998

15.2

16.4

5.2

33.7

0.7

48

AOD9604

100.9907

32.4

11.9

17.8

3.7

1.8

64

AOD9604

(7–16)

met.

50.9888

37.7

22.4

11.9

10.5

0.6

54

Buserelin

50.9970

11.6

4.5

14.5

20.9

055

Deslorelin

50.9952

11.8

4.0

7.1

12.1

0.1

55

Desmopressin

100.9935

13.6

23.3

21.6

7.3

067

Felypressin

0.5

0.9990

14.7

6.3

3.6

25.5

0.7

86

Fertirelin

10.9993

14.5

14.5

18.5

22.1

0.1

69

GHRP-1

100.9919

21.8

17.4

12.7

11.2

3.6

142

GHRP-1

(3–6)met.

50.9991

18.4

53.4

8.9

14.9

0.1

48

GHRP-2

20.9991

13.3

5.2

6.8

11.2

0.1

55

GHRP-2

(1–3)met.

20.9998

17.5

7.8

5.3

25.1

0.1

76

GHRP-3

50.9973

19.5

2.7

27.8

27.4

0.1

79

GHRP-4

20.9996

15.7

14.7

2.6

28.2

053

GHRP-5

20.9964

16.8

14.6

4.8

22.4

033

GHRP-6

20.9975

7.9

15.2

25.6

41.6

9.1

96

GHRP-6

(2–5)met.

20.9982

24.7

31.6

10.6

19.8

0.1

52

Gly-A

lexamorelin

50.9990

16.9

11.5

18.7

13.8

8.7

117

Gly-G

HRP-1

20.9949

12.3

18.8

5.8

19.4

4.2

156

Gly-G

HRP-2

100.9988

7.8

11.9

16.0

17.4

5.3

55

Gly-G

HRP-3

50.9965

18.9

4.7

21.0

24.6

0.1

77

Gly-G

HRP-4

10.9993

14.5

17.4

4.0

26.1

052

Gly-G

HRP-5

50.9972

12.2

19.9

6.8

12.8

0.1

45

Gly-G

HRP-6

20.9987

18.1

15.7

7.8

17.8

7.8

135

Gly-H

exarelin

20.9986

15.4

13.4

13.9

13.0

6.4

123

Gly-Ipamorelin

10.9984

10.0

12.6

9.9

69.6

7.8

125

Goserelin

20.9965

8.7

10.8

10.9

17.3

0.1

57

Hexarelin

100.9999

19.7

15.1

12.3

14.6

5.7

84

Hexarelin

(1–3)met.

0.5

0.9958

25.7

14.9

5.6

24.4

6.9

51

Histrelin

50.9980

18.3

15.5

14.7

20.8

1.5

62

Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide...

variation of this method. A time-dependent degradation of thecompounds could not be observed.

Post-administration samples

It is desirable to show that the substances cannot only bedetected in fortified samples but also in authentic specimens,for example, collected in the course of administration studies.Since serum samples from a previous elimination study withGHRP-2 and GHRP-6 were available, DBS with a Hct = 40%were reconstructed. As shown in Fig. 2, both GHRP-2 andGHRP-6 could be unambiguously identified by their charac-teristic target and confirming ions in DBS generated fromblood samples reconstructed with serum that was collectedup to 90 min after application.

In DBS generated from blood reconstituted with serum thatwas collected 4.5 h post-administration, specific signals werestill detectable but could not be confirmed by a second isoto-pic peak (confirming signal). A blank sample showed no in-terfering signals at the respective retention times. A standardcalibration curve for GHRP-2 and GHRP-6 between 5 and50 ng/mL was applied to estimate the concentration from theDBS samples, yielding levels between 2 and 10 ng/mL withinthe detection window (30–90 min). The results correspondwith the concentrations of GHRP-2 and GHRP-6 after a singlei.v. injection from other studies [19, 20]. It should be notedhere that serum blood levels are expected to be slightly differ-ent from full blood levels because the analytes will most likelynot be found at identical concentrations in red blood cells andserum. Nevertheless, the applicability of the testing procedureto post-administration samples could be successfullydemonstrated.

Hct determination by NIR spectroscopy

The NIR spectroscope was used for non-destructive Hct mea-surements of DBS to obtain preliminary data in a pilot projectonly. A previously developed NIR model [28] was adopted todetermine the Hct, and the influence of storage time and tem-perature on the Hct measurements was studied. For this pur-pose, EDTA-stabilized venous blood from one volunteer wasutilized, and regular measurements in triplicate (spots 1–3)were performed over a period of 3 weeks while storing DBScards in the dark at RT, 4 °C, and − 20 °C. DBS cards weredried for 6 h at RT and then stored under the conditions de-scribed above. A reference value of 38% measured by aSysmex XN-1000 analyzer was determined on the first dayafter blood drawing. Regardless of the storage time, slightdifferences in a temperature-dependent manner in the rangeof 32.0–38.7% were observed. The DBS cards stored for atleast 1 day at RT showed Hct values close to the referencevalue while cards stored at 4 °C and − 20 °C resulted in lowervalues as visualized in Fig. 3. In spite of sealing the cards inT

able2

(contin

ued)

Com

pound

LOD

[ng/mL]

(n=6)

Linearity

(R)

LOD-100

ng/m

L(n=1)

Precision

at20

ng/m

L[%

](n=6)

Precision

at50

ng/m

L[%

](n=6)

Precision

at100ng/m

L[%

](n=6)

Recoveryat

20ng/m

L[%

](n=6)

Carryover

after

100ng/m

L[%

](n=1)

Matrix

effects

[%](n=3)

Ibutam

oren

10.9999

24.9

15.5

13.2

46.3

0.4

46

Ipam

orelin

0.5

0.9989

13.5

17.9

7.1

50.9

9.9

76

Ipam

orelin

(1–4)met.

0.5

0.9966

19.6

20.1

7.8

33.8

3.4

68

Lecirelin

(dalmarelin)

0.5

0.9992

11.1

3.4

13.9

24.2

054

Leuprolide

0.5

0.9985

10.3

5.3

10.5

27.5

056

Leuprolide(1–3)met.

10.9981

23.1

16.7

11.5

23.6

0.1

75

LHRH

20.9992

14.4

4.7

24.0

21.4

0.2

70

Nafarelin

100.9862

13.8

4.0

8.3

8.0

0.2

49

Nafarelin

(5–10)

met.

20.9948

19.8

15.9

9.5

11.5

0.1

57

Peforelin

200.9985

26.6

12.8

14.0

34.9

0.5

61

Tabimorelin

100.9991

23.3

10.6

8.1

46.4

051

TB500

10.9996

22.2

12.8

6.7

19.5

1.6

54

Triptorelin

100.9961

15.5

3.5

7.3

14.0

056

T. Lange et al.

plastic bags with a desiccant, the differences in temperature orhumidity might influence the molecular vibrations of the DBSmatrix that are crucial for the NIR spectrum calculation. Atime-dependent change in total hemoglobin was not assumed

as others have already shown its stability in DBS [36]. Aconsiderably slower drying or freezing of remaining moistureof the DBS matrix at reduced temperatures would be in accor-dance with this observation, suggesting a prolonged drying

3.2 3.6 4.0 4.4 4.8 5.2 5.6 6.0 6.4

Time [min]

0

100

0

100

0

100

0

100

0

100

0

100

0

100

0

100 3.63

4.89

5.03

5.27

5.46

5.65

5.78

6.03

NL: 1.46E6

m/z= 445.2531

NL: 1.59E6

m/z= 427.2088

NL: 7.49E6

m/z= 528.7231

NL: 3.42E5

m/z= 521.7077

NL: 8.84E5

m/z= 630.2889

NL: 2.94E6

m/z= 453.1881

NL: 1.01E6

m/z= 356.7001

NL: 2.77E5

m/z= 655.4038

8.2 8.6 9.0 9.4 9.8 10.2

Time [min]

0

100

0

100

0

100

0

100

0

100

0

100

0

100

0

1008.56

8.77

8.91

8.94

9.33

9.74

9.75

NL: 1.17E6

m/z= 641.8276

NL: 6.66E5

m/z= 401.2242

NL: 3.55E5

m/z= 409.7210

NL: 2.48E6

m/z= 875.4563

NL: 3.95E5

m/z= 661.8251

NL: 9.28E6

m/z= 612.3231

NL: 2.26E6

m/z= 608.2980

5.8 6.2 6.6 7.0 7.4

Time [min]

0

100

0

100

0

100

0

100

0

100

0

100

0

100

0

1006.18

6.25

6.39

6.80

6.83

6.83

6.92

6.93

NL: 8.87E5

m/z= 520.7257

NL: 4.40E5

m/z= 712.4253

NL: 1.45E6

m/z= 591.7938

NL: 9.66E5

m/z= 385.2108

NL: 3.70E5

m/z= 437.2296

NL: 4.51E6

m/z= 577.2987

NL: 8.94E5

m/z= 662.3409

NL: 6.42E5

m/z= 465.7403

10.0 10.4 10.8 11.2

Time [min]

0

100

0

100

0

100

0

100

0

100

0

100

0

100

0

100

9.79

10.21

10.39

10.37

10.32

10.48

10.53

11.03

NL: 2.10E6

m/z= 665.3194

NL: 2.05E6

m/z= 609.2820

NL: 1.56E7

m/z= 529.2479

NL: 2.09E6

m/z= 623.2957

NL: 3.94E5

m/z= 529.3173

NL: 2.68E6

m/z= 771.3613

NL: 9.85E5

m/z= 828.3828

NL: 1.13E6

m/z= 620.2883

NL: 8.17E6

m/z= 547.3391

7.4 7.8 8.2 8.6

Time [min]

0

100

0

100

0

100

0

100

0

100

0

100

0

100

0

1007.69

7.70

7.73

7.76

8.16 8.40

8.17

8.22

NL: 3.03E6

m/z= 361.1950

NL: 1.85E6

m/z= 358.1761

NL: 6.72E5

m/z= 506.7612

NL: 3.53E6

m/z= 585.2820

NL: 2.09E6

m/z= 605.3300

NL: 9.31E5

m/z= 635.3280

NL: 4.01E5

m/z= 656.3227

TB500

Hexarelin Met

[Lys8]-Vasopressin, ISTD 1

AOD9604 7-16 Met

Peforelin

Leuprolide 1-3 Met

Ipamorelin

GHRP-3

Deslorelin

Nafarelin Met

Gly-GHRP-2

GHRP-2

Nafarelin

d4-Ala-GHRP-4, ISTD 3

GHRP-4

9.6

Felypressin

Gly-GHRP-3

LHRH

Gly-Ipamorelin

GHRP-6

Fertirelin

Histrelin

Gly-GHRP-6

Gly-GHRP-4

GHRP-6 Met

Ibutamoren

Alexamorelin Met

Tabimorelin

GHRP-5

Gly-GHRP-5

GHRP-1 Met

d3-Ala-GHRP-2 Met, ISTD 2

GHRP-2 Met

Gly-GHRP-1

Ipamorelin Met

Leuprolide (left)

Lecirelin (right)

Goserelin

Triptorelin

6.6 7.0 7.4 7.8

Time [min]

0

100

0

100

0

100

0

100

0

100

0

100

0

100

0

100 6.96

7.00

7.00

7.02

7.04

7.10

7.43

7.66

NL: 1.60E5

m/z= 444.2374

NL: 4.67E5

m/z= 472.7481

NL: 2.21E5

m/z= 907.9375

NL: 1.38E6

m/z= 584.3065

NL: 1.54E5

m/z= 1069.4342

NL: 6.24E5

m/z= 508.2667

NL: 9.16E5

m/z= 479.7560

NL: 1.96E5

m/z= 478.2505

Hexarelin

Gly-Hexarelin

AOD9604

Alarelin

Desmopressin

Gly-Alexamorelin

Alexamorelin

GHRP-1

8.50 NL: 1.62E6

m/z= 620.3353Buserelin

11.09

Anamorelin

7.3

Fig. 1 Extracted ion chromatograms (mass tolerance ± 5 ppm) of asample from a female volunteer obtained by a finger prick. The samplewas either analyzed as blank (dashed line) or with 20 ng/mL of a peptide

mix containing all 46 target compounds. The rows of the 3 ISTDs are alsoshown at their respective retention time

Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide...

phase (1–2 days at RT followed by storage at RT with desic-cant) for a reliable Hct measurement using the presented ap-proach with DBS cards.

Since all measurements on the NIR spectroscope byOostendorp et al. were based on DBS prepared from venousEDTA-stabilized blood [28], the applicability of the Hct cal-culation to capillary DBS collected by finger prick was

investigated. Therefore, the Hct of different authentic DBSsamples collected from ten volunteers was determined. DBSwere sealed and dried for 2 days at RT. The measured Hctwere found to be considerably lower than expected with arange from 24 to 40% (and an average of 31%), potentiallyresulting from different confounding factors. In some cases,finger blood collection was complicated by a slow blood flow,

7.4 7.6 7.8 8.0 8.2

50

1007.69 NL: 1.30E6

m/z = 361.1970

6.8 7.0 7.2

Time [min]

50

100

50

100 NL: 0

m/z = 437.2333

NL: 0

m/z= 437.7312

9.6 9.8 10.0 10.2

50

1009.74 NL: 3.36E6

m/z = 612.3268

7.4 7.6 7.8 8.0 8.2

50

1007.68 NL: 5.55E5

m/z = 361.1972

8.8 9.0 9.2 9.4

Time [min]

50

100

50

100

Rela

tive A

bundance [%

]

8.89

8.89

NL: 1.93E4

m/z = 409.7242

NL: 2.50E3

m/z = 410.7265

6.8 7.0 7.2

Time [min]

50

100

50

1006.86

6.86

NL: 7.01E3

m/z = 437.2333

NL: 2.84E3

m/z = 437.7312

9.6 9.8 10.0 10.2

50

1009.74 NL: 5.99E6

m/z = 612.3268

8.8 9.0 9.2 9.4

Time [min]

50

100

50

100

Rela

tive A

bundance [%

]

NL: 0

m/z = 409.7242

NL: 0

m/z = 410.7265

9.4

8.6 6.4 6.6

9.4

8.6 6.4 6.6

blank

s.c. (post-administration)

GHRP-2

confirming

GHRP-2

target

GHRP-2

confirming

GHRP-2

target

GHRP-6

confirming

GHRP-6

target

GHRP-6

confirming

GHRP-6

target

d3-GHRP-2 met.

(ISTD)

d3-GHRP-2 met.

(ISTD)

d4-GHRP-4

(ISTD)

d4-GHRP-4

(ISTD)

Fig. 2 Extracted ionchromatograms (mass tolerance±10 ppm) of a blank sample and as.c. post-administration sampleshowing signals of GHRP-2 andGHRP-6

Fig. 3 The influence of storagetime and temperature on the Hctmeasurements from DBS by NIRspectroscopywas studied. SeveralDBS cards were stored over aperiod of 3 weeks at RT, 4 °C, and− 20 °C and samples weremeasured in triplicates (spots 1–3). The error bars result from thestandard deviations of therespective experiments

T. Lange et al.

and even a slight squeezing of the finger can provoke exudateleaking into the collected blood causing a dilution of the sam-ple. In addition, Hct values reportedly differ between bodyregions [37] and variation in capillary density, cutaneousblood content and red blood cell velocity must be taken intoaccount [38]. The influence of anticoagulant (K2EDTA) onthe NIR spectra might necessitate further investigations aswell, suggesting more comprehensive studies in order to en-able a holistic classification of Hct values obtained from NIRspectroscopy. As shown before, the different Hct values hadno effect on the detection of the substances in this qualitativeassay. However, the Hct is relevant in case of quantitativeanalyses regarding threshold substances determined fromDBS, since the conversion from full blood into plasma con-centrations seems to be decisive for the determination of con-centration thresholds. A Hct-dependent correction factorcould overcome this previous limitation when using DBS.

DBS sampling methods

Both DBS sampling methods either from the arm or from thefinger were successfully validated regarding selectivity.Compared with DBS collection from the finger prick, mostvolunteers described the arm device to be virtually painlessand more comfortable to use. It is noteworthy that samplecollection failure was reduced compared with the procedurewith the finger lancet. The “TAP” devices could be leveragedto refine the method of blood collection.

Conclusion

In sports drug testing, the demand for a higher samplethroughput is continuously increasing. DBS sample collectionmay contribute to this development. The complementary ma-trix is mainly characterized by its cost efficiency (in terms ofstorage and shipping) and minimal invasiveness. In order todeter doping, a fully automated robotic DBS sample prepara-tion with LC-HRMS detection was developed. The multi-analyte initial testing approach comprises 46 lower molecularmass peptide or non-peptide (mimetic) target analytes <2 kDaof different receptor agonist categories such as agonists thatbind to ghrelin receptors, GnRH receptors, hGh receptors, andADH receptors. Due to the discovery of glycine-modifiedanalogues, the list of analytes was extended preventively witha series of nine glycine-modified peptides, mainly GHRPs. Inaddition, GHRP-1 that could not be detected in urine before[10], and felypressin, an ADH receptor agonist, were imple-mented for the first time in an anti-doping detection proce-dure. The vast majority of the drug candidates are still underdevelopment, in clinical trials or were discontinued. To thebest of our knowledge, leuprolide, felypressin, LHRH,histrelin, desmopressin, GHRP-2, goserelin, triptorelin,

buserelin, and nafarelin have obtained clinical approval. Forveterinary use only, the application for marketing authoriza-tion was concerned for peforelin, alarelin, lecirelin, anddeslorelin. Independent from the state of development, allthese pharmaceuticals are available on the black market andpose a potential risk in relation to doping practices.

Despite the small DBS volume of 20 μL, sensitivities en-abling the detection of an illicit use were achieved.Remarkably, more than 60% of the analytes could be detectedbelow the WADA’s minimum required performance limit(MRPL) of 2 ng/mL for urine [39]. Until now, no MRPLspecification is available for serum/plasma or DBS.

Furthermore, an upstream NIR spectroscope for non-destructive Hct measurement was implemented and the as-say’s robustness in terms of extractability was demonstratedfor different Hct values. This approach could contribute to aHct-dependent correction and would support quantitativeDBS applications in the future.

As a proof of concept, artificial DBS samples obtainedfrom post-administration specimens containing GHRP-2 andGHRP-6 were successfully analyzed.

The automated DBS preparation of 6 samples lasted approx-imately 2 h, as long as the subsequent LC-HRMS analysis andwas therefore ideally suited for a just-in-time workflow. Theautomation and the possibility of a programmable nesting ofthe preparation steps within the sequence would allow for anincreased sample throughput compared with sophisticatedmanual sample preparation. Finally, if desired, the entire assaycould be easily extended with new compounds.

Funding information Open Access funding provided by Projekt DEAL.The study was funded by the Federal Ministry of the Interior, Buildingand Community of the Federal Republic of Germany (Berlin, Germany),the Manfred-Donike Institute for Doping Analysis (Cologne, Germany),Anti-Doping Switzerland (Berne, Switzerland), theNational Anti-DopingAgency (Bonn, Germany), and the World Anti-Doping Agency(Montreal, Canada, grant #ISF17A12MT).

Compliance with ethical standards All procedures per-formed in studies involving human participants were in accordance withthe ethical standards of the institutional research committee and with the1964 Helsinki declaration and its later amendments. Ethical approval wasobtained from the local ethics committee of the German Sport UniversityCologne (#022/2017) and informed consent was provided by all partici-pants included in the study.

Conflict of interest The authors declare that they have no conflict ofinterest.

Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in any medium or format, aslong as you give appropriate credit to the original author(s) and thesource, provide a link to the Creative Commons licence, and indicate ifchanges weremade. The images or other third party material in this articleare included in the article's Creative Commons licence, unless indicatedotherwise in a credit line to the material. If material is not included in thearticle's Creative Commons licence and your intended use is not

Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide...

permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view acopy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

References

1. Okano M, Sato M, Ikekita A, Kageyama S. Determination ofgrowth hormone secretagogue pralmorelin (GHRP-2) and its me-tabolite in human urine by liquid chromatography/electrospray ion-ization tandem mass spectrometry. Rapid Commun MassSpectrom. 2010;24(14):2046–56. https://doi.org/10.1002/rcm.4619.

2. Thomas A, Kohler M, Mester J, Geyer H, Schänzer W, Petrou M,et al. Identification of the growth-hormone-releasing peptide-2(GHRP-2) in a nutritional supplement. Drug Test Anal. 2010;2(3):144–8. https://doi.org/10.1002/dta.120.

3. Thomas A, Höppner S, Geyer H, Schänzer W, Petrou M,Kwiatkowska D, et al. Determination of growth hormone releasingpeptides (GHRP) and their major metabolites in human urine fordoping controls by means of liquid chromatography mass spec-trometry. Anal Bioanal Chem. 2011;401(2):507–16. https://doi.org/10.1007/s00216-011-4702-3.

4. Thomas A, Solymos E, Schänzer W, Baume N, Saugy M, DellannaF, et al. Determination of vasopressin and desmopressin in urine bymeans of liquid chromatography coupled to quadrupole time-of-flight mass spectrometry for doping control purposes. Anal ChimActa. 2011;707(1–2):107–13. https://doi.org/10.1016/j.aca.2011.09.027.

5. Pinyot A, Nikolovski Z, Bosch J, Such-Sanmartin G, Kageyama S,Segura J, et al. Growth hormone secretagogues: out of competition.Anal Bioanal Chem. 2012;402(3):1101–8. https://doi.org/10.1007/s00216-011-5544-8.

6. Thomas A, Walpurgis K, Krug O, Schänzer W, Thevis M.Determination of prohibited, small peptides in urine for sports drugtesting by means of nano-liquid chromatography/benchtop quadru-pole orbitrap tandem-mass spectrometry. J Chromatogr A.2012;1259:251–7. https://doi.org/10.1016/j.chroma.2012.07.022.

7. Timms M, Hall N, Levina V, Vine J, Steel R. A high-throughputLC-MS/MS screen for GHRP in equine and human urine, featuringpeptide derivatization for improved chromatography. Drug TestAnal. 2014;6(10):985–95. https://doi.org/10.1002/dta.1624.

8. Cox HD, Hughes CM, Eichner D. Detection of GHRP-2 andGHRP-6 in urine samples from athletes. Drug Test Anal.2015;7(5):439–44. https://doi.org/10.1002/dta.1791.

9. Semenistaya E, Zvereva I, Thomas A, Thevis M, Krotov G,Rodchenkov G. Determination of growth hormone releasing pep-tides metabolites in human urine after nasal administration ofGHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin. DrugTest Anal. 2015;7(10):919–25. https://doi.org/10.1002/dta.1787.

10. Görgens C, Guddat S, Thomas A, Thevis M. Recent improvementsin sports drug testing concerning the initial testing for peptidic drugs(< 2 kDa) - sample preparation, mass spectrometric detection, anddata review. Drug Test Anal. 2018. https://doi.org/10.1002/dta.2503.

11. Thomas A, Görgens C, Guddat S, Thieme D, Dellanna F, SchänzerW, et al. Simplifying and expanding the screening for peptides<2 kDa by direct urine injection, liquid chromatography, and ionmobility mass spectrometry. J Sep Sci. 2016;39(2):333–41. https://doi.org/10.1002/jssc.201501060.

12. Judak P, Grainger J, Goebel C, Van Eenoo P, Deventer K. DMSOassisted electrospray ionization for the detection of small peptidehormones in urine by dilute-and-shoot-liquid-chromatography-high

resolution mass spectrometry. J Am Soc Mass Spectrom.2017;28(8):1657–65. https://doi.org/10.1007/s13361-017-1670-7.

13. Handelsman DJ. Performance enhancing hormone doping in sport.In: Feingold KR, Anawalt B, Boyce A, Chrousos G, Dungan K,Grossman A et al., editors. Endotext. South Dartmouth (MA)MDText.com, Inc.; 2015.

14. WADA. Anti-Doping Testing Figures. 2017. https://www.wada-ama.org/en/resources/laboratories/anti-doping-testing-figures-report. Accessed 12/07/2018.

15. Poplawska M, Blazewicz A. Identification of a novel growth hor-mone releasing peptide (a glycine analogue of GHRP-2) in a seizedinjection vial. Drug Test Anal. 2018. https://doi.org/10.1002/dta.2467.

16. Krug O, Thomas A, Malerod-Fjeld H, Dehnes Y, Laussmann T,Feldmann I, et al. Analysis of new growth promoting black marketproducts. Growth Hormon IGF Res. 2018;41:1–6. https://doi.org/10.1016/j.ghir.2018.05.001.

17. Gajda PM, Holm NB, Hoej LJ, Rasmussen BS, Dalsgaard PW,Reitzel LA, et al. Glycine-modified growth hormone secretagoguesidentified in seized doping material. Drug Test Anal. 2018. https://doi.org/10.1002/dta.2489.

18. WADA. The World Anti-Doping Code. International Standard.Prohibited List January 2020. https://www.wada-ama.org/sites/default/files/wada_2020_english_prohibited_list_0.pdf. Accessed07/02/2020.

19. Pihoker C, Kearns GL, French D, Bowers CY. Pharmacokineticsand pharmacodynamics of growth hormone-releasing peptide-2: Aphase I study in children. J Clin Endocrinol Metab. 1998;83(4):1168–72. https://doi.org/10.1210/jc.83.4.1168.

20. Cabrales A, Gil J, Fernandez E, Valenzuela C, Hernandez F, GarciaI, et al. Pharmacokinetic study of growth hormone-releasing peptide6 (GHRP-6) in nine male healthy volunteers. Eur J Pharm Sci.2013;48(1–2):40–6. https://doi.org/10.1016/j.ejps.2012.10.006.

21. Ferro P, Krotov G, Zvereva I, Perez-Mana C, Mateus J, Segura J.Detection of growth hormone releasing peptides in serum by acompetitive receptor binding assay. J Chromatogr. 2017;8(1).https://doi.org/10.4172/2157-7064.1000351.

22. Ferro P, Krotov G, Zvereva I, Rodchenkov G, Segura J. Structure-activity relationship for peptidic growth hormone secretagogues.Drug Test Anal. 2017;9(1):87–95. https://doi.org/10.1002/dta.1947.

23. Thomas A, Delahaut P, Krug O, Schänzer W, Thevis M.Metabolism of growth hormone releasing peptides. Anal Chem.2012;84(23):10252–9. https://doi.org/10.1021/ac302034w.

24. Dib J, Tretzel L, Piper T, Logajda A, Kühne D, Schänzer W, ThevisM. Screening for adiponectin receptor agonists and their metabo-lites in urine and dried blood spots. Clin Mass Spectrom.2017;6(13–20).

25. Tretzel L, Thomas A, Piper T, Hedeland M, Geyer H, Schänzer W,et al. Fully automated determination of nicotine and its major me-tabolites in whole blood by means of a DBS online-SPE LC-HR-MS/MS approach for sports drug testing. J Pharm Biomed Anal.2016;123:132–40. https://doi.org/10.1016/j.jpba.2016.02.009.

26. Lehmann S, Delaby C, Vialaret J, Ducos J, Hirtz C. Current andfuture use of “dried blood spot” analyses in clinical chemistry. ClinChem Lab Med. 2013;51(10):1897–909. https://doi.org/10.1515/cclm-2013-0228.

27. O'MaraM, Hudson-Curtis B, Olson K, Yueh Y, Dunn J, Spooner N.The effect of hematocrit and punch location on assay bias duringquantitative bioanalysis of dried blood spot samples. Bioanalysis.2011;3(20):2335–47. https://doi.org/10.4155/bio.11.220.

28. Oostendorp M, El Amrani M, Diemel EC, Hekman D, vanMaarseveen EM. Measurement of hematocrit in dried blood spotsusing near-infrared spectroscopy: robust, fast, and nondestructive.Clin Chem. 2016;62(11):1534–6. https://doi.org/10.1373/clinchem.2016.263053.

T. Lange et al.

29. Blicharz TM, Gong P, Bunner BM, Chu LL, Leonard KM,Wakefield JA, et al. Microneedle-based device for the one-steppainless collection of capillary blood samples. Nat Biomed Eng.2018;2(3):151–7. https://doi.org/10.1038/s41551-018-0194-1.

30. Knoop A, Thomas A, Fichant E, Delahaut P, Schanzer W, ThevisM. Qualitative identification of growth hormone-releasing hor-mones in human plasma by means of immunoaffinity purificationand LC-HRMS/MS. Anal Bioanal Chem. 2016;408(12):3145–53.https://doi.org/10.1007/s00216-016-9377-3.

31. WADA. The World Anti-Doping Code. International Standard forLaboratories January. 2019. https://www.wada-ama.org/sites/default/files/resources/files/isl_nov2019.pdf. Accessed 25/03/2020.

32. Hahne H, Pachl F, Ruprecht B, Maier SK, Klaeger S, Helm D, et al.DMSO enhances electrospray response, boosting sensitivity of pro-teomic experiments. Nat Methods. 2013;10(10):989–91. https://doi.org/10.1038/nmeth.2610.

33. Meyer JG, A Komives E. Charge state coalescence duringelectrospray ionization improves peptide identification by tandemmass spectrometry. J Am Soc Mass Spectrom. 2012;23(8):1390–9.https://doi.org/10.1007/s13361-012-0404-0.

34. Zvereva I, Semenistaya E, Krotov G, Rodchenkov G. Comparisonof various in vitro model systems of the metabolism of syntheticdoping peptides: proteolytic enzymes, human blood serum, liverand kidney microsomes and liver S9 fraction. J Proteome.2016;149:85–97. https://doi.org/10.1016/j.jprot.2016.08.016.

35. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies forthe assessment of matrix effect in quantitative bioanalyticalmethods based on HPLC-MS/MS. Anal Chem. 2003;75(13):3019–30.

36. Capiau S, Wilk LS, Aalders MC, Stove CP. A novel, nondestruc-tive, dried blood spot-based hematocrit prediction method usingnoncontact diffuse reflectance spectroscopy. Anal Chem.2016;88(12):6538–46. https://doi.org/10.1021/acs.analchem.6b01321.

37. McHedlishvili G, Varazashvili M, Kumsishvili T, Lobjanidze I.Regional hematocrit changes related to blood flow conditions inthe arterial bed. Clin Hemorheol Microcirc. 2003;29(2):71–9.

38. Simonen P, O'Brien M, Hamilton C, Ashcroft J, Denham J. Normalvariation in cutaneous blood content and red blood cell velocity inhumans. Physiol Meas. 1997;18(3):155–70. https://doi.org/10.1088/0967-3334/18/3/001.

39. WADA. WADA Technical Document - Minimum RequiredPerformance Levels of Non-Threshold Substances. 2019. https://www.wada-ama.org/sites/default/files/resources/files/td2019mrpl_eng.pdf. Accessed 25/03/2020.

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Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide...